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Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation.

Tummala P, Mali RS, Guzman E, Zhang X, Mitton KP - Mol. Vis. (2010)

Bottom Line: Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation.Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI).Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene's promoter complex.

View Article: PubMed Central - PubMed

Affiliation: Eye Research Institute, Oakland University, Rochester, MI 48309-4401, USA.

ABSTRACT

Purpose: During retinal development, post-mitotic neural progenitor cells must activate thousands of genes to complete synaptogenesis and terminal maturation. While many of these genes are known, others remain beyond the sensitivity of expression microarray analysis. Some of these elusive gene activation events can be detected by mapping changes in RNA polymerase-II (Pol-II) association around transcription start sites.

Methods: High-resolution (35 bp) chromatin immunoprecipitation (ChIP)-on-chip was used to map changes in Pol-II binding surrounding 26,000 gene transcription start sites during photoreceptor maturation of the mouse neural retina, comparing postnatal age 25 (P25) to P2. Coverage was 10-12 kb per transcription start site, including 2.5 kb downstream. Pol-II-active regions were mapped to the mouse genomic DNA sequence by using computational methods (Tiling Analysis Software-TAS program), and the ratio of maximum Pol-II binding (P25/P2) was calculated for each gene. A validation set of 36 genes (3%), representing a full range of Pol-II signal ratios (P25/P2), were examined with quantitative ChIP assays for transcriptionally active Pol-II. Gene expression assays were also performed for 19 genes of the validation set, again on independent samples. FLT-3 Interacting Zinc-finger-1 (FIZ1), a zinc-finger protein that associates with active promoter complexes of photoreceptor-specific genes, provided an additional ChIP marker to highlight genes activated in the mature neural retina. To demonstrate the use of ChIP-on-chip predictions to find novel gene activation events, four additional genes were selected for quantitative PCR analysis (qRT-PCR analysis); these four genes have human homologs located in unidentified retinal disease regions: Solute carrier family 25 member 33 (Slc25a33), Lysophosphatidylcholine acyltransferase 1 (Lpcat1), Coiled-coil domain-containing 126 (Ccdc126), and ADP-ribosylation factor-like 4D (Arl4d).

Results: ChIP-on-chip Pol-II peak signal ratios >1.8 predicted increased amounts of transcribing Pol-II and increased expression with an estimated 97% accuracy, based on analysis of the validation gene set. Using this threshold ratio, 1,101 genes were predicted to experience increased binding of Pol-II in their promoter regions during terminal maturation of the neural retina. Over 800 of these gene activations were additional to those previously reported by microarray analysis. Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation. Expression of all four genes was diminished in adult retinas lacking rod photoreceptors (Rd1 mice) compared to normal retinas (90% loss for Ccdc126 and Arl4d). For rhodopsin (Rho), a marker of photoreceptor maturation, two regions of maximum Pol-II signal corresponded to the upstream rhodopsin enhancer region and the rhodopsin proximal promoter region.

Conclusions: High-resolution maps of Pol-II binding around transcription start sites were generated for the postnatal mouse retina; which can predict activation increases for a specific gene of interest. Novel gene activation predictions are enriched for biologic functions relevant to vision, neural function, and chromatin regulation. Use of the data set to detect novel activation increases was demonstrated by expression analysis for several genes that have human homologs located within unidentified retinal disease regions: Slc25a33, Lpcat1, Ccdc126, and Arl4d. Analysis of photoreceptor-deficient retinas indicated that all four genes are expressed in photoreceptors. Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI). Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene's promoter complex.

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Related in: MedlinePlus

Expression changes (mRNA) of evaluation genes. A-C: Relative mRNA concentrations in the mouse neural retina were measured by qPCR (real time) for the developmental ages P2 and P25. Concentrations were normalized to the beta-Actin mRNA concentration. Bars indicate standard deviation for triplicate assays. Taqman chemistry was used for target specificity with hydrolysis probes that span exon junctions. Rho, Rcvn, Pde6b, and Sag1 are key markers of photoreceptor-specific gene expression. Genes are grouped to account for different scales of relative expression. Most genes had Pol-II peak signal ratios > 1.8, as determined from temporal Pol-II ChIP-on-Chip analysis, except for: Hdac9 (ratio 1.6), Hdac10 (ratio 1.7), and Grik2 (ratio 0.9, Table 1). Genes: Rhodopsin (Rho), Recoverin (Rcvrn), Retinoschisis 1 (Rs1), Phosphodiesterase 6b (Pde6b), S-antigen (Sag), Elongation of very long chain fatty acids-like 4 (Elovl4), D4, zinc and double PHD fingers, family 3 (Dpf3), Spectrin repeat containing, nuclear envelope 1 (Syne1), Solute carrier family 38, Na/H -coupled glutamine transporter, member 3 (Slc38a3), Family with sequence similarity 53, member B (A930008G19Rik, Fam53b), Nuclear receptor subfamily 1, group D, member 1 (Nr1d1), Jumonji domain containing 2C (Jmjd2c), Histone deacetylase 9 (Hdac9), Bardet-Biedl syndrome 9 (E130103I17Rik, Bbs9), Histone deacetylase 10 (Hdac10), Glutamate receptor, ionotropic, kainate 2 (beta 2) (Grik2), Solute carrier family 24, Na/K/Ca exchanger, member 1 (Slc24a1).
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f3: Expression changes (mRNA) of evaluation genes. A-C: Relative mRNA concentrations in the mouse neural retina were measured by qPCR (real time) for the developmental ages P2 and P25. Concentrations were normalized to the beta-Actin mRNA concentration. Bars indicate standard deviation for triplicate assays. Taqman chemistry was used for target specificity with hydrolysis probes that span exon junctions. Rho, Rcvn, Pde6b, and Sag1 are key markers of photoreceptor-specific gene expression. Genes are grouped to account for different scales of relative expression. Most genes had Pol-II peak signal ratios > 1.8, as determined from temporal Pol-II ChIP-on-Chip analysis, except for: Hdac9 (ratio 1.6), Hdac10 (ratio 1.7), and Grik2 (ratio 0.9, Table 1). Genes: Rhodopsin (Rho), Recoverin (Rcvrn), Retinoschisis 1 (Rs1), Phosphodiesterase 6b (Pde6b), S-antigen (Sag), Elongation of very long chain fatty acids-like 4 (Elovl4), D4, zinc and double PHD fingers, family 3 (Dpf3), Spectrin repeat containing, nuclear envelope 1 (Syne1), Solute carrier family 38, Na/H -coupled glutamine transporter, member 3 (Slc38a3), Family with sequence similarity 53, member B (A930008G19Rik, Fam53b), Nuclear receptor subfamily 1, group D, member 1 (Nr1d1), Jumonji domain containing 2C (Jmjd2c), Histone deacetylase 9 (Hdac9), Bardet-Biedl syndrome 9 (E130103I17Rik, Bbs9), Histone deacetylase 10 (Hdac10), Glutamate receptor, ionotropic, kainate 2 (beta 2) (Grik2), Solute carrier family 24, Na/K/Ca exchanger, member 1 (Slc24a1).

Mentions: For further evaluation, gene expression assays were completed for 19 genes from the validation gene set, which also represented a full range of Pol-II peak signal ratios. These results are graphed in Figure 3. All of the test genes with Pol-II peak signal ratios >1.8 showed increased expression at P25 based upon neural retina mRNA concentration. Based on the Pol-II Q-ChIP and gene expression results, we concluded that a ChIP-on-chip Pol-II peak signal ratio (P25/P2) ≥1.8 predicts significant increases in the gene activation state with >97% accuracy. This ratio was used for subsequent prediction analysis from the full Pol-II ChIP-on-chip data set.


Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation.

Tummala P, Mali RS, Guzman E, Zhang X, Mitton KP - Mol. Vis. (2010)

Expression changes (mRNA) of evaluation genes. A-C: Relative mRNA concentrations in the mouse neural retina were measured by qPCR (real time) for the developmental ages P2 and P25. Concentrations were normalized to the beta-Actin mRNA concentration. Bars indicate standard deviation for triplicate assays. Taqman chemistry was used for target specificity with hydrolysis probes that span exon junctions. Rho, Rcvn, Pde6b, and Sag1 are key markers of photoreceptor-specific gene expression. Genes are grouped to account for different scales of relative expression. Most genes had Pol-II peak signal ratios > 1.8, as determined from temporal Pol-II ChIP-on-Chip analysis, except for: Hdac9 (ratio 1.6), Hdac10 (ratio 1.7), and Grik2 (ratio 0.9, Table 1). Genes: Rhodopsin (Rho), Recoverin (Rcvrn), Retinoschisis 1 (Rs1), Phosphodiesterase 6b (Pde6b), S-antigen (Sag), Elongation of very long chain fatty acids-like 4 (Elovl4), D4, zinc and double PHD fingers, family 3 (Dpf3), Spectrin repeat containing, nuclear envelope 1 (Syne1), Solute carrier family 38, Na/H -coupled glutamine transporter, member 3 (Slc38a3), Family with sequence similarity 53, member B (A930008G19Rik, Fam53b), Nuclear receptor subfamily 1, group D, member 1 (Nr1d1), Jumonji domain containing 2C (Jmjd2c), Histone deacetylase 9 (Hdac9), Bardet-Biedl syndrome 9 (E130103I17Rik, Bbs9), Histone deacetylase 10 (Hdac10), Glutamate receptor, ionotropic, kainate 2 (beta 2) (Grik2), Solute carrier family 24, Na/K/Ca exchanger, member 1 (Slc24a1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2822553&req=5

f3: Expression changes (mRNA) of evaluation genes. A-C: Relative mRNA concentrations in the mouse neural retina were measured by qPCR (real time) for the developmental ages P2 and P25. Concentrations were normalized to the beta-Actin mRNA concentration. Bars indicate standard deviation for triplicate assays. Taqman chemistry was used for target specificity with hydrolysis probes that span exon junctions. Rho, Rcvn, Pde6b, and Sag1 are key markers of photoreceptor-specific gene expression. Genes are grouped to account for different scales of relative expression. Most genes had Pol-II peak signal ratios > 1.8, as determined from temporal Pol-II ChIP-on-Chip analysis, except for: Hdac9 (ratio 1.6), Hdac10 (ratio 1.7), and Grik2 (ratio 0.9, Table 1). Genes: Rhodopsin (Rho), Recoverin (Rcvrn), Retinoschisis 1 (Rs1), Phosphodiesterase 6b (Pde6b), S-antigen (Sag), Elongation of very long chain fatty acids-like 4 (Elovl4), D4, zinc and double PHD fingers, family 3 (Dpf3), Spectrin repeat containing, nuclear envelope 1 (Syne1), Solute carrier family 38, Na/H -coupled glutamine transporter, member 3 (Slc38a3), Family with sequence similarity 53, member B (A930008G19Rik, Fam53b), Nuclear receptor subfamily 1, group D, member 1 (Nr1d1), Jumonji domain containing 2C (Jmjd2c), Histone deacetylase 9 (Hdac9), Bardet-Biedl syndrome 9 (E130103I17Rik, Bbs9), Histone deacetylase 10 (Hdac10), Glutamate receptor, ionotropic, kainate 2 (beta 2) (Grik2), Solute carrier family 24, Na/K/Ca exchanger, member 1 (Slc24a1).
Mentions: For further evaluation, gene expression assays were completed for 19 genes from the validation gene set, which also represented a full range of Pol-II peak signal ratios. These results are graphed in Figure 3. All of the test genes with Pol-II peak signal ratios >1.8 showed increased expression at P25 based upon neural retina mRNA concentration. Based on the Pol-II Q-ChIP and gene expression results, we concluded that a ChIP-on-chip Pol-II peak signal ratio (P25/P2) ≥1.8 predicts significant increases in the gene activation state with >97% accuracy. This ratio was used for subsequent prediction analysis from the full Pol-II ChIP-on-chip data set.

Bottom Line: Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation.Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI).Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene's promoter complex.

View Article: PubMed Central - PubMed

Affiliation: Eye Research Institute, Oakland University, Rochester, MI 48309-4401, USA.

ABSTRACT

Purpose: During retinal development, post-mitotic neural progenitor cells must activate thousands of genes to complete synaptogenesis and terminal maturation. While many of these genes are known, others remain beyond the sensitivity of expression microarray analysis. Some of these elusive gene activation events can be detected by mapping changes in RNA polymerase-II (Pol-II) association around transcription start sites.

Methods: High-resolution (35 bp) chromatin immunoprecipitation (ChIP)-on-chip was used to map changes in Pol-II binding surrounding 26,000 gene transcription start sites during photoreceptor maturation of the mouse neural retina, comparing postnatal age 25 (P25) to P2. Coverage was 10-12 kb per transcription start site, including 2.5 kb downstream. Pol-II-active regions were mapped to the mouse genomic DNA sequence by using computational methods (Tiling Analysis Software-TAS program), and the ratio of maximum Pol-II binding (P25/P2) was calculated for each gene. A validation set of 36 genes (3%), representing a full range of Pol-II signal ratios (P25/P2), were examined with quantitative ChIP assays for transcriptionally active Pol-II. Gene expression assays were also performed for 19 genes of the validation set, again on independent samples. FLT-3 Interacting Zinc-finger-1 (FIZ1), a zinc-finger protein that associates with active promoter complexes of photoreceptor-specific genes, provided an additional ChIP marker to highlight genes activated in the mature neural retina. To demonstrate the use of ChIP-on-chip predictions to find novel gene activation events, four additional genes were selected for quantitative PCR analysis (qRT-PCR analysis); these four genes have human homologs located in unidentified retinal disease regions: Solute carrier family 25 member 33 (Slc25a33), Lysophosphatidylcholine acyltransferase 1 (Lpcat1), Coiled-coil domain-containing 126 (Ccdc126), and ADP-ribosylation factor-like 4D (Arl4d).

Results: ChIP-on-chip Pol-II peak signal ratios >1.8 predicted increased amounts of transcribing Pol-II and increased expression with an estimated 97% accuracy, based on analysis of the validation gene set. Using this threshold ratio, 1,101 genes were predicted to experience increased binding of Pol-II in their promoter regions during terminal maturation of the neural retina. Over 800 of these gene activations were additional to those previously reported by microarray analysis. Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation. Expression of all four genes was diminished in adult retinas lacking rod photoreceptors (Rd1 mice) compared to normal retinas (90% loss for Ccdc126 and Arl4d). For rhodopsin (Rho), a marker of photoreceptor maturation, two regions of maximum Pol-II signal corresponded to the upstream rhodopsin enhancer region and the rhodopsin proximal promoter region.

Conclusions: High-resolution maps of Pol-II binding around transcription start sites were generated for the postnatal mouse retina; which can predict activation increases for a specific gene of interest. Novel gene activation predictions are enriched for biologic functions relevant to vision, neural function, and chromatin regulation. Use of the data set to detect novel activation increases was demonstrated by expression analysis for several genes that have human homologs located within unidentified retinal disease regions: Slc25a33, Lpcat1, Ccdc126, and Arl4d. Analysis of photoreceptor-deficient retinas indicated that all four genes are expressed in photoreceptors. Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI). Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene's promoter complex.

Show MeSH
Related in: MedlinePlus